Function Study Of BpSPL2-BpPIF4 In Adventitious Root Development And Abiotic Stress Of Betula Platyphylla Suk.

SPLs（SQUAMOSA PROMOTER BINDING PROTEIN-LIKE）are plant-specific transcription factors which play diverse roles in plant development,the role of this transcription factor in abiotic stress has been widely reported.In this study,Betula platyphylla was used as material to study the role of BpSPL2 in salt stress,preliminarily verified the relationship between BpSPL2 and Bp PIF4,genetic transformation of Bp PIF4 was performed and analysed the function of Bp PIF4 gene.1.Analyzed the expression patterns of BpSPLs family genes under salt,drought stress and ABA treatment,and found that BpSPLs expressions were affected by high salt,drought stress and ABA treatment,but the expression pattern of each gene was different,suggested that BpSPLs might be involved in stress response and play different roles.2.The phenotypes of BpSPL2 transgenic plants which obtained in the early stage were observed.It was found that inhibition of BpSPL2 expression increased plant height,leaf area,node spacing and the color of leaves got lighter.For the underground part,the number of adventitious roots and dry weight of the plants 35S::BpSPL2-SRDX increased significantly,which root system was more developed.3.NBT（Nitrotetrazolium Blue Chloride）staining was performed on salt-treated tissue culture seedlings,compared with the wild type,the overexpression plants had darker blue leaves and more O^2-content.After salt stress treatment,overexpression of BpSPL2 was the most damaged,followed by wild type and inhibition expression was the least damaged. Physiological indicators showed that the activity of SOD,POD and proline content of 35S::BpSPL2-SRDX were significantly higher than those of 35S::BpSPL2 and WT.q RT-PCR results showed that after 192 h salt treatment,genes related to reactive oxygen,osmotic regulation substances and gibberellin metabolism in stem base of 35S::BpSPL2-SRDX were significantly higher than 35S::BpSPL2 and wild-type birch in the same period.In conclusion,BpSPL2 negatively regulated the resistance of Betula platyphylla to salt stress.4.The regulatory relationship between BpSPL2 and Bp PIF4 was preliminarily verified by yeast one-hybrid assay,BpSPL2 regulates the expression of downstream gene Bp PIF4.Cloned Bp PIF4 gene from birch,which was 1443 bp,encoded 480 amino acids.Bioinformatics analysis showed that Bp PIF4 protein was acidic,unstable and hydrophilic.The secondary structure of the protein was irregular curly,and it contained four low-complexity regions and an HLH domain.Subcellular localization showed that Bp PIF4 was located in the nucleus.q RT-PCR analysis showed that the expression of Bp PIF4 gene was tissue-specific,most abundant in petiole,and could be affected by high salt,drought stress and ABA treatment.Bp PIF4 overexpression and inhibition of Betula platyphylla were successfully transformed by leaf disc method,12 overexpression lines and 5 inhibition lines were obtained.It was found that the adventitious roots of 35S::Bp PIF4-SRDX got decreased number and shortened length,while the root length of overexpression plants increased by17.69%compared with that of wild type.These results indicated that Bp PIF4 was involved in the regulation of adventitious root formation of birch.